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C. elegans FOG-3/Tob can either promote or inhibit germline proliferation, depending on gene dosage and genetic context.

Snow JJ, Lee MH, Verheyden J, Kroll-Conner PL, Kimble J - Oncogene (2012)

Bottom Line: Thus, fog-3 mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 alleles.Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations.A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore, this FOG-3 tumor-promoting effect is context dependent.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT
Vertebrate Tob/BTG proteins inhibit cell proliferation when overexpressed in tissue-culture cells, and they can function as tumor suppressors in mice. The single Caenorhabditis elegans Tob/BTG ortholog, FOG-3, by contrast, was identified from its loss-of-function phenotype as a regulator of sperm fate specification. Here we report that FOG-3 also regulates proliferation in the germline tissue. We first demonstrate that FOG-3 is a positive regulator of germline proliferation. Thus, fog-3 mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 alleles. A similar decrease also occurred in fog-3/+ heterozygotes, again for both fog-3 alleles, revealing a haplo-insufficient effect on proliferation. Therefore, FOG-3 normally promotes proliferation, and two copies of the fog-3 gene are required for this function. We next overexpressed FOG-3 by removal of FBF, the collective term for FBF-1 and FBF-2, two nearly identical PUF RNA-binding proteins. We find that overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants; whereas germ cells stop dividing and instead differentiate in fbf-1 fbf-2 double mutants, they continue to proliferate in fog-3; fbf-1 fbf-2 triple mutants. Therefore, like its vertebrate Tob/BTG cousins, overexpressed FOG-3 is 'antiproliferative'. Indeed, some fog-3; fbf-1 fbf-2 mutants possess small tumors, suggesting that FOG-3 can act as a tumor suppressor. Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations. A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore, this FOG-3 tumor-promoting effect is context dependent. We conclude that FOG-3 can either promote or inhibit proliferation in a manner that is sensitive to both genetic context and gene dosage. The discovery of these FOG-3 effects on proliferation has implications for our understanding of vertebrate Tob/BTG proteins and their influence on normal development and tumorigenesis.

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The undetectable level of FOG-3::FLAG in the mitotic zone is dependent upon FBF. (a–d) Dissected gonads from early L4 hermaphrodites harboring a FLAG-tagged FOG-3 transgene to rescue their lack of endogenous fog-3; (e) schematic of gonad in (a,b); (f) schematic of gonad in (c,d). Gonads are outlined and germline regions marked: mitotic zone (MZ), transition zone (TZ) and pachytene region (PR). Most MZ germ cells are in the mitotic cell cycle; all TZ and PR germ cells have entered the meiotic cell cycle with TZ cells in early meiotic prophase and PR germ cells progressing through the pachytene stage of meiotic prophase. See Kimble and Crittenden (17) for details. Asterisk indicates gonadal distal end. (a,c) Staining with anti-FLAG antibody to visualize transgenic FOG-3::FLAG rescuing protein; (b,d) DAPI staining to visualize all nuclei and visualize boundaries of germline regions. Widefield images were taken at same magnification with the same settings; scale bar in (a) represents 50 μM and serves for all images. (a,b) FOG-3::FLAG is not detectable in the mitotic zone (MZ) in a genetic strain with wild-type FBF. (c,d) FOG-3::FLAG becomes easily detectable in the proximal part of the MZ in a genetic strain lacking FBF. The images shown are representative for this dramatic strain difference in the MZ. We note that this image also shows a staining difference in the proximal part of the pachytene region, which was not quantitated and is beyond the scope of this study. (e,f) Positions at which FOG-3::FLAG staining was quantitated using ImageJ: black square, distal MZ; grey square, proximal MZ; and open square, distal PR; (g) ImageJ quantitation of FOG-3::FLAG staining, including both average pixel intensity and range.
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Figure 2: The undetectable level of FOG-3::FLAG in the mitotic zone is dependent upon FBF. (a–d) Dissected gonads from early L4 hermaphrodites harboring a FLAG-tagged FOG-3 transgene to rescue their lack of endogenous fog-3; (e) schematic of gonad in (a,b); (f) schematic of gonad in (c,d). Gonads are outlined and germline regions marked: mitotic zone (MZ), transition zone (TZ) and pachytene region (PR). Most MZ germ cells are in the mitotic cell cycle; all TZ and PR germ cells have entered the meiotic cell cycle with TZ cells in early meiotic prophase and PR germ cells progressing through the pachytene stage of meiotic prophase. See Kimble and Crittenden (17) for details. Asterisk indicates gonadal distal end. (a,c) Staining with anti-FLAG antibody to visualize transgenic FOG-3::FLAG rescuing protein; (b,d) DAPI staining to visualize all nuclei and visualize boundaries of germline regions. Widefield images were taken at same magnification with the same settings; scale bar in (a) represents 50 μM and serves for all images. (a,b) FOG-3::FLAG is not detectable in the mitotic zone (MZ) in a genetic strain with wild-type FBF. (c,d) FOG-3::FLAG becomes easily detectable in the proximal part of the MZ in a genetic strain lacking FBF. The images shown are representative for this dramatic strain difference in the MZ. We note that this image also shows a staining difference in the proximal part of the pachytene region, which was not quantitated and is beyond the scope of this study. (e,f) Positions at which FOG-3::FLAG staining was quantitated using ImageJ: black square, distal MZ; grey square, proximal MZ; and open square, distal PR; (g) ImageJ quantitation of FOG-3::FLAG staining, including both average pixel intensity and range.

Mentions: Normally FBF is most abundant in mitotically-dividing germ cells at the distal end of the gonad (21, 24), and FOG-3::FLAG is most abundant more proximally in germ cells that have left the mitotic zone and entered meiotic prophase (23). We therefore focused on the mitotic zone, the region of actively dividing germ cells, to ask if FBF affects FOG-3::FLAG expression. Confirming previous results (23), FOG-3::FLAG was not detected in the mitotic zone of a germline harboring wild-type FBF (Figures 2a and b). By contrast, in an fbf-1 fbf-2 double mutant germline with no FBF, FOG-3::FLAG became easily detectable in the proximal mitotic zone (Figures 2c and d). Quantitation with ImageJ confirmed this FOG-3::FLAG increase (Figures 2e and f). We do not know if this FBF effect is direct or indirect. Nonetheless, FBF clearly affects FOG-3::FLAG expression in the mitotic zone.


C. elegans FOG-3/Tob can either promote or inhibit germline proliferation, depending on gene dosage and genetic context.

Snow JJ, Lee MH, Verheyden J, Kroll-Conner PL, Kimble J - Oncogene (2012)

The undetectable level of FOG-3::FLAG in the mitotic zone is dependent upon FBF. (a–d) Dissected gonads from early L4 hermaphrodites harboring a FLAG-tagged FOG-3 transgene to rescue their lack of endogenous fog-3; (e) schematic of gonad in (a,b); (f) schematic of gonad in (c,d). Gonads are outlined and germline regions marked: mitotic zone (MZ), transition zone (TZ) and pachytene region (PR). Most MZ germ cells are in the mitotic cell cycle; all TZ and PR germ cells have entered the meiotic cell cycle with TZ cells in early meiotic prophase and PR germ cells progressing through the pachytene stage of meiotic prophase. See Kimble and Crittenden (17) for details. Asterisk indicates gonadal distal end. (a,c) Staining with anti-FLAG antibody to visualize transgenic FOG-3::FLAG rescuing protein; (b,d) DAPI staining to visualize all nuclei and visualize boundaries of germline regions. Widefield images were taken at same magnification with the same settings; scale bar in (a) represents 50 μM and serves for all images. (a,b) FOG-3::FLAG is not detectable in the mitotic zone (MZ) in a genetic strain with wild-type FBF. (c,d) FOG-3::FLAG becomes easily detectable in the proximal part of the MZ in a genetic strain lacking FBF. The images shown are representative for this dramatic strain difference in the MZ. We note that this image also shows a staining difference in the proximal part of the pachytene region, which was not quantitated and is beyond the scope of this study. (e,f) Positions at which FOG-3::FLAG staining was quantitated using ImageJ: black square, distal MZ; grey square, proximal MZ; and open square, distal PR; (g) ImageJ quantitation of FOG-3::FLAG staining, including both average pixel intensity and range.
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Related In: Results  -  Collection

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Figure 2: The undetectable level of FOG-3::FLAG in the mitotic zone is dependent upon FBF. (a–d) Dissected gonads from early L4 hermaphrodites harboring a FLAG-tagged FOG-3 transgene to rescue their lack of endogenous fog-3; (e) schematic of gonad in (a,b); (f) schematic of gonad in (c,d). Gonads are outlined and germline regions marked: mitotic zone (MZ), transition zone (TZ) and pachytene region (PR). Most MZ germ cells are in the mitotic cell cycle; all TZ and PR germ cells have entered the meiotic cell cycle with TZ cells in early meiotic prophase and PR germ cells progressing through the pachytene stage of meiotic prophase. See Kimble and Crittenden (17) for details. Asterisk indicates gonadal distal end. (a,c) Staining with anti-FLAG antibody to visualize transgenic FOG-3::FLAG rescuing protein; (b,d) DAPI staining to visualize all nuclei and visualize boundaries of germline regions. Widefield images were taken at same magnification with the same settings; scale bar in (a) represents 50 μM and serves for all images. (a,b) FOG-3::FLAG is not detectable in the mitotic zone (MZ) in a genetic strain with wild-type FBF. (c,d) FOG-3::FLAG becomes easily detectable in the proximal part of the MZ in a genetic strain lacking FBF. The images shown are representative for this dramatic strain difference in the MZ. We note that this image also shows a staining difference in the proximal part of the pachytene region, which was not quantitated and is beyond the scope of this study. (e,f) Positions at which FOG-3::FLAG staining was quantitated using ImageJ: black square, distal MZ; grey square, proximal MZ; and open square, distal PR; (g) ImageJ quantitation of FOG-3::FLAG staining, including both average pixel intensity and range.
Mentions: Normally FBF is most abundant in mitotically-dividing germ cells at the distal end of the gonad (21, 24), and FOG-3::FLAG is most abundant more proximally in germ cells that have left the mitotic zone and entered meiotic prophase (23). We therefore focused on the mitotic zone, the region of actively dividing germ cells, to ask if FBF affects FOG-3::FLAG expression. Confirming previous results (23), FOG-3::FLAG was not detected in the mitotic zone of a germline harboring wild-type FBF (Figures 2a and b). By contrast, in an fbf-1 fbf-2 double mutant germline with no FBF, FOG-3::FLAG became easily detectable in the proximal mitotic zone (Figures 2c and d). Quantitation with ImageJ confirmed this FOG-3::FLAG increase (Figures 2e and f). We do not know if this FBF effect is direct or indirect. Nonetheless, FBF clearly affects FOG-3::FLAG expression in the mitotic zone.

Bottom Line: Thus, fog-3 mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 alleles.Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations.A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore, this FOG-3 tumor-promoting effect is context dependent.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT
Vertebrate Tob/BTG proteins inhibit cell proliferation when overexpressed in tissue-culture cells, and they can function as tumor suppressors in mice. The single Caenorhabditis elegans Tob/BTG ortholog, FOG-3, by contrast, was identified from its loss-of-function phenotype as a regulator of sperm fate specification. Here we report that FOG-3 also regulates proliferation in the germline tissue. We first demonstrate that FOG-3 is a positive regulator of germline proliferation. Thus, fog-3 mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 alleles. A similar decrease also occurred in fog-3/+ heterozygotes, again for both fog-3 alleles, revealing a haplo-insufficient effect on proliferation. Therefore, FOG-3 normally promotes proliferation, and two copies of the fog-3 gene are required for this function. We next overexpressed FOG-3 by removal of FBF, the collective term for FBF-1 and FBF-2, two nearly identical PUF RNA-binding proteins. We find that overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants; whereas germ cells stop dividing and instead differentiate in fbf-1 fbf-2 double mutants, they continue to proliferate in fog-3; fbf-1 fbf-2 triple mutants. Therefore, like its vertebrate Tob/BTG cousins, overexpressed FOG-3 is 'antiproliferative'. Indeed, some fog-3; fbf-1 fbf-2 mutants possess small tumors, suggesting that FOG-3 can act as a tumor suppressor. Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations. A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore, this FOG-3 tumor-promoting effect is context dependent. We conclude that FOG-3 can either promote or inhibit proliferation in a manner that is sensitive to both genetic context and gene dosage. The discovery of these FOG-3 effects on proliferation has implications for our understanding of vertebrate Tob/BTG proteins and their influence on normal development and tumorigenesis.

Show MeSH
Related in: MedlinePlus